Systems Biology in Reproductive Medicine, 2013, 59: 223–226
Copyright © 2013 Informa Healthcare USA, Inc.
ISSN 1939-6368 print/1939-6376 online
DOI: 10.3109/19396368.2013.787129
APPLICATION NOTES
Anterior positioning of sex chromosomes on the head of human sperm
sorted using visible wavelengths
Sofia Alçada-Morais1,3, Ana
Paula Sousa1,2, Artur Paiva3, Teresa Almeida-Santos2,4, and
∗
João Ramalho-Santos1,5
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1
Biology of Reproduction and Stem Cell Group, CNC- Center for Neuroscience and Cell Biology, University of Coimbra, 2Human
Reproduction Service, University Hospitals of Coimbra, 3Histocompatibilty Center, 4Faculty of Medicine, University of Coimbra,
5
Department of Life Sciences, University of Coimbra, Coimbra, Portugal
The human ejaculate contains subpopulations of sperm with
distinct properties. Human X- and Y-bearing sperm were separated with fluorescence activated cell sorting. To avoid the use
of UV light the quantitative DNA dyes DRAQ5® and Dyecycle™
Vybrant® Violet were used. Sorting efficiency was similar for
both dyes, but lower than what is usually obtained with the
classical method involving Hoechst 33342 and UV light (6070% enrichment, versus 80-90%). A total of 2,739 spermatozoa
were evaluated, from seven distinct samples using fluorescence
in situ hybridization (FISH) chromosomal probes. No differences
were found in sorted and unsorted populations in terms
of chromosome positioning, and numeric chromosomal
anomalies were not more evident following cell sorting.
Furthermore in both sorted and unsorted populations the sex
chromosomes were clearly located in the anterior portion of
the sperm head, while a control autosome (chromosome 18)
showed no such tendency, confirming previous findings.
These results suggest that other quantitative DNA dyes may
be used for sex chromosome-based human sperm sorting,
but with lower efficiency than the standard UV-Hoechst
based assay.
production, or clinical purposes, such as avoiding the transmission of X-linked diseases [Cran and Johnson 1996;
Garner 2006; Johnson et al. 1993; Sills et al. 1998].
However, the most effective protocol to efficiently separate
these subpopulations is using quantitative DNA dyes and
flow cytometry coupled to cell sorting [Cran and Johnson
1996; Garner 2006; 2009; Seidel 2012]. This methodology
relies on the different size/DNA content of the sex chromosomes (and thus on distinct DNA content in X- and
Y-bearing spermatozoa), and its efficiency in different mammalian species therefore depends on the relative size difference of the sex chromosomes [Cran and Johnson 1996;
Garner 2006; Sills et al. 1998]. This difference is quite
modest in humans (about 3%), leading to controversial
results, but also to a few reported successes, which may
have ethical implications in terms of possible sex-selection
[Fugger et al. 1998; Sills et al. 1998; Vidal et al. 1998].
Despite its successful use in many species, a frequent point
of contention is the common use of the UV-excited DNA
dye Hoechst 33342 for sperm separation based on DNA
content [Garner 2009].
Interestingly it has been well established that chromosome positioning in mammalian sperm in general, and of
sex chromosomes in particular, is non-random. Although
the nucleus of the male gamete is less studied and understood than nuclei from interphase somatic cells, it is believed
that chromosome positions may have a physiological significance in terms of early transcriptional activity in the embryo,
and possible consequences for male infertility or in the use of
assisted reproduction technologies [Ioannou and Griffin
2011; Zalensky and Zalenskaya 2007]. Chromosome positions in the mammalian sperm head may vary in different
ways, both in terms of where the chromosome tends to be
predominantly located (in the anterior, equatorial, or posterior regions of the head, more centrally, or more peripherally) or where certain chromosome structures (centromeres,
Keywords cell sorting, DNA dyes, human, sex chromosomes,
sperm
Introduction
The human ejaculate contains subpopulations of sperm with
distinct properties, and functional abilities [Sousa et al.
2011]. Two obvious subpopulations are those defined by
the sex chromosomes, theoretically dividing an ejaculate in
half, with 50% of Y- and 50% of X- bearing cells. These subpopulations may have distinct biochemical properties,
besides a clear role in embryonic sex determination.
Indeed, many different methodologies have been described
to separate X and Y-bearing sperm for study, animal
Received 17 December 2012; accepted 06 February 2013.
∗
Address correspondence to João Ramalho-Santos, Department of Life Sciences, University of Coimbra, PO Box 3046, 3001-401 Coimbra, Portugal.
E-mail: [email protected]
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S. Alçada-Morais et al.
is well below what is usually reported using the HoechstUV based approach, suggesting that this method remains
the standard for this purpose, with consistent efficiency in
the 80-90% range [Garner 2006; 2009; Seidel 2012]. Thus
the dyes tested here may not be particularly useful for practical purposes. Furthermore, these results precluded more
detailed functional and biochemical analysis of X- and Ybearing sperm subpopulations, as enrichment was clearly
not sufficient to ensure reliable results. It should be noted
that human sperm are more difficult to separate than
sperm from other species due to the small difference in
DNA content, and this may hamper the search for alternative methodologies [Garner 2006; Johnson et al. 1993].
Given the nature of the assay, however, we were able to
perform fluorescence in situ hybridization (FISH) analysis
on the sorted subpopulations. No statistically significant
differences in terms of chromosome position were observed
when sorted samples were compared with unsorted samples
using probes for the X and Y chromosomes, suggesting that
sex chromosome position does not influence DNA contentbased sorting (data not shown). Also no obvious numerical
chromosomal anomalies were more prevalent in sorted subpopulations (data not shown). However a clear anterior distribution of both sex chromosomes was evident, while
chromosome 18 showed no such tendency (Fig. 2). This
was especially clear for the X chromosome, and is in accordance with previous data for this chromosome [Hazzouri et al.
2000; Luetjens et al. 1999]. This has been confirmed more
recently for the Y chromosome, albeit in this case anterior
positioning occurs to a lesser degree [Millan et al. 2012].
Interestingly, the position of X and Y chromosomes relative
telomeres) tend to reside [Finch et al. 2008; Foster et al. 2005;
Haaf and Ward 1995; Hazzouri et al. 2000; Luetjens et al.
1999; Manvelyan et al. 2008; Millan et al. 2012; Mudrak
et al. 2005; Olszewska et al. 2008; Tilgen et al. 2001; Zalenskaya and Zalensky 2004].
Specific attention has been paid to the position of the sex
chromosomes in the human sperm head (specifically the X
chromosome), with several early studies using multiple
samples pointing to its preferential location on the anterior
portion of the sperm head [Hazzouri et al. 2000; Luetjens
et al. 1999].
In this work we separated human sperm cells by flow
cytometry and cell sorting using DNA content to study
specific populations enriched in Y- and X- bearing chromosomes. To circumvent the use of UV light, we have used the
quantitative DNA dyes DRAQ5® and Dyecycle™ Vybrant®
Violet, which are excited at visible (i.e., higher) wavelengths
[Mari et al. 2010; Zhao et al. 2009].
Results and Discussion
We attempted to find alternatives to the classically used
Hoechst 33342 and UV light for sex chromosome-based
sperm sorting. DRAQ5® and Dyecycle™ Vybrant® Violet
yielded statistically identical results (data not shown). No
clear separate peaks in terms of DNA content were ever
visible, and thus we sorted two populations on the edges
of the main peak (Fig. 1). This resulted in a modest but significant 60-70% enrichment of a specific sex chromosomebearing sperm, rather than the 50% random distribution
found in non-sorted cells ( p < 0.05; Fig. 1). However this
®
Figure 1. Separating X and Y- bearing human sperm. When separating human sperm according to DNA content using Dyecycle™ Vybrant Violet
a strongly labeled sperm population was visible (A, elipse), but when samples were scanned according to fluorescence intensity only one peak was
visible, showing no distinct subpopulations clearly definable by DNA content (B). If the extremes of this peak were gated (defined in B) sperm subpopulations could be obtained enriched in either Y (green spots) or X (red spots) bearing chromosomes, depending on whether the ascending (lower
DNA content; C) or descending (higher DNA content; D) arm of the peak was selected, respectively. Panels C and D are representative FISH images,
with DAPI (blue) used as a nuclear counterstain. Results with DRAQ5 were essentially identical (data not shown).
®
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Sex chromosome-based sperm sorting
225
Figure 2. Chromosome positions in sorted human sperm. Human sperm were sorted according to DNA content and analyzed by fluorescence in
situ hybridization (FISH) using probes for chromosomes X, Y, and 18. For localization purposes, and as shown in an example in the top right corner,
the sperm head was divided into three equal quadrants (anterior, equatorial, posterior), with the tail insertion (asterisk) used as a landmark for the
posterior side, and the localization of the FISH spot (arrow) classified accordingly. A total of 2,739 spermatozoa were evaluated, from seven distinct
samples, and percentages calculated for each sample. Error bars represent standard deviation. ∗ statistically significant differences ( p < 0.05)
to the sperm head and to other chromosomes do not always
coincide, suggesting distinct behaviors of sex chromosomes.
This is perhaps related to their distinct transcriptional activities in the early embryo and in somatic interphase nuclei
[Millan et al. 2012; Zalenskaya and Zalensky 2004]. This
specific positioning may be relevant when using intracytoplasmic sperm injection (ICSI) given that in this case
sperm head decondensation may be delayed in the anterior
portion, due to the persistence in this region within the remnants of sperm structures (the acrosome, the perinuclear
theca) that are normally discarded during fertilization
[Hewitson et al. 1999; Ramalho-Santos et al. 2000].
Materials and Methods
Samples
Patients undergoing routine semen analysis or fertility treatment were recruited from the Fertility Clinic (University Hospitals of Coimbra, Portugal). All patients signed informed
consent forms, and all human material was used in accordance with the appropriate ethical and Internal Review Board
(IRB) guidelines provided by the University Hospitals of
Coimbra. Fresh semen samples were obtained by masturbation after 3 to 5 d of sexual abstinence and semen analysis
was carried out in conformity to the World Health Organization Guidelines [WHO 2010]. Semen samples were prepared
by density gradient centrifugation as described elsewhere
[Amaral et al. 2007; Sousa et al. 2011]. All samples used
were obtained from healthy individuals and were normozoospermic for concentration (i.e., ≥ 15 million sperm/mL), motility (i.e., ≥ 40% motile sperm), and morphology (i.e., ≥ 4%
normal forms), and had no leukocytes (or any other round
Copyright © 2013 Informa Healthcare USA, Inc.
cells) after semen preparation or other obvious indications
of health problems that might affect sperm quality.
DNA dyes and cell sorting
Live sperm suspensions (20 million of sperm/mL) were incubated with 10 µM of DRAQ5® (BioStatus Limited, UK) or 9
µM of Dyecycle™ Vybrant® Violet (Molecular Probes,
Eugene, OR, USA), for 5 min at room temperature and 30
min at 37°C, respectively. The sorting process was carried
out with a BD FACSAria™ cell-sorting system (BD FACSAria
III; BD Biosciences, NJ, USA) at 635 nm (DRAQ5® ) and
407 nm (Dyecycle™ Vybrant® Violet ) wavelength with the
following settings: laser power – 13 mW, nozzle – 70 µm,
and sort setup – medium. After separation, the sperm cell
suspensions were dropped onto clean glass slides, air dried,
and kept at −20°C until used.
Fluorescence in situ hybridization (FISH)
FISH analysis was carried out with modifications from previous protocols [Almeida Santos et al. 2002; Ramalho-Santos
et al. 2004]. Probes used were CEP X red and CEP Y green
(Vysis, Abbott Laboratories, IL, USA). ILM or CEP Y
green and CEP 18 red (Vysis). Slides were counterstained
with 10 µL DAPI in antifade solution (VectaShield mounting medium, Vector Labs, Burlingame, CA, USA) and observed using a Zeiss Axiophot II microscope (Carl Zeiss,
Göttingen, Germany) equipped with a triple band pass filter.
A total of 2,739 spermatozoa were evaluated, from seven distinct samples. For localization purposes the sperm head was
divided into three equal quadrants (anterior, equatorial, posterior), with the tail insertion functioning as a landmark for
the posterior side, as described previously [Luetjens et al. 1999].
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S. Alçada-Morais et al.
Statistical analysis
Statistical analysis was performed using the IBM SPSS® 20
software (Chicago, IL, USA). All variables were checked
for normal distribution through the one-sample Kolmogorov-Smirnov test. One-way ANOVA was used to compare the
results obtained in the three regions considered and the posthoc analyses were done using Tukey’s test. Statistical significance was considered when p < 0.05.
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Acknowledgments
All lab members are thanked for helpful discussions. This
work was done as part of the Masters Program in Cell and
Molecular Biology at the Department of Life Sciences,
University of Coimbra (SAM).
Declaration of interests: The authors have no interests to
declare.
Author contributions: Defined the project, and finalized the
manuscript: JRS; Processed sperm samples for analysis: APS,
TAS; Performed all FISH experiments, as well as flow cytometry
data collection and initial analysis: SAM; Performed flow cytometry analysis: AP; All authors analyzed the data; Wrote the
paper: JRS, APS, SAM; All authors approved the paper.
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